Project Summary A fundamental question in cell biology is how cells build functionally and structurally distinct microtubule (MT) networks using a seemingly simple set of protein building blocks -- ??-tubulin heterodimers. For many years, the cytoskeletal field has focused on the roles of MT-binding proteins and motors as the primary regulators of network structure and function. However, it is now clear that the ??-tubulin building blocks are not so simple. Rather than a uniform track, the MT surface is a molecularly diverse landscape that is generated by genetic and posttranslational differences between ??-tubulins. The `tubulin code' model posits that changes to the intrinsically disordered carboxy-terminal tail (CTT) domains of ??-tubulins create a molecular code at the MT surface that is ?read? by MT-binding proteins. The overarching goals of this proposal are to establish mechanistic connections between CTTs and the conformational diversity of MT ends and lattices, and to understand how this regulates complex MT network functions at the cellular level. This structure proposal features and function, and a multi-system, multi-scale approach to understanding how how these lead to changes in the function of MT networks CTTs impact tubulin in cells. My lab has established expertise in investigating tubulin using approaches that integrate genetic models, live-cell imaging and protein biochemistry. Our progress over the past five years has furthered our understanding of how ??- tubulin CTTs regulate MT networks, and, more broadly, how tubulin heterogeneity impacts cellular and developmental processes. The proposed project will build upon our expertise to expand the current model of the tubulin code and give broader insights into mechanisms of MT function. Our goals for the next five years include 1) define how CTTs guide the structure of MT ends to regulate MT dynamics, 2) determine how blends of ??-tubulins with different amino acid sequences and posttranslational modifications give rise to complex behaviors at the level of MT networks in cells, 3) define how CTTs promote the directionality of kinesin motility along MTs, and 4) establish a novel role for tubulins in buffering intracellular cation concentrations. Our synergistic approach is uniquely suited to advance knowledge of tubulin structure and function that will be important in a broad range of contexts, provide new insights into how microtubule networks regulate and respond to changes at the level of tubulin subunits, and how these impact different cellular contexts.